Folprp4 Gene Involved in the Conidiogenesis and Mycelial Growth in Fusarium oxysporum f. sp. lini

doi:10.13523/j.cb.2110024

China Biotechnology

2022, Vol. 42

Issue (3): 13-26 DOI: 10.13523/j.cb.2110024

Folprp4 Gene Involved in the Conidiogenesis and Mycelial Growth in Fusarium oxysporum f. sp. lini

DONG Hui-xia,HOU Zhan-ming^*(

)

College of Life Science and Technology, Inner Mongolia Normal University, Key Laboratory of Biodiversity Conservation and Sustainable Utilization, Advanced Universities of Inner Mongolia, Hohhot 010022, China

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Abstract

Objective: To identify the Folprp4 gene in Fusarium oxysporum f. sp. lini and reveal its function and pathogenic correlation in Fusarium oxysporum f. sp. lini. Methods: Based on the principle of homologous recombination, the Split-Marker strategy was applied to construct the gene deletion cassette containing hygromycin resistance gene (hph) according to the genomic sequence of Folprp4 gene determined. The constructs were transformed into wild-type protoplasts mediated by PEG and the transformants were screened on TCC medium containing hygromycin B. The Folprp4 gene deletion mutants were confirmed by PCR with positive and negative primers. To do a complement test, the vector pZDH1 containing Folprp4 gene was constructed, and transformed into the deletion mutants. Results: Compared with wild type (hm) and ectopic insertion mutant (ecFolprp4), the growth of the mycelia of the deletion mutants was seriously obstructed and its colony looked like a tiny dot when the wild type and ectopic insertion mutant extended full of the plate. Another striking change of the deletion mutant was that the conidial production of ΔFolprp4 were significantly decreased. The infection assay showed that the virulence of ΔFolprp4 on flax seedlings was significantly reduced. The complement test indicated that the transformants of the complementary vector (Folprp4-C) recovered in colony morphology, growth rate, conidial yield and virulence of wild-type strain. Conclusion: The results revealed that Folprp4 gene was tightly associated with mycelial growth, conidial formation and pathogenicity in Fusarium oxysporum f. sp. lini.

Key words： Folprp4 Fusarium oxysporum f. sp. lini Split-Marker Gene deletion Pathogenicity

Received: 14 October 2021 Published: 07 April 2022

ZTFLH:

Q819

Corresponding Authors: Zhan-ming HOU E-mail: houzhm@imnu.edu.cn

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Cite this article:

DONG Hui-xia, HOU Zhan-ming. Folprp4 Gene Involved in the Conidiogenesis and Mycelial Growth in Fusarium oxysporum f. sp. lini. China Biotechnology, 2022, 42(3): 13-26.

URL:

https://manu60.magtech.com.cn/biotech/10.13523/j.cb.2110024 OR https://manu60.magtech.com.cn/biotech/Y2022/V42/I3/13

Table 1 This experiment involves important primers

Fig.1 Amino acid sequence alignment of four fungus

Fig.2 Acquisition of Folprp4 gene DNA and cDNA sequence M: 1 kb plus DNA Ladder (a) Fusarium oxysporum Schl. f. sp. lini total genomic DNA (b) Fusarium oxysporum Schl. f. sp. lini total RNA (c~i) Using hm as template, PCR products were amplified with Folprp4 G1F-G2R; Folprp4 G3F-G4R ; Folprp4 G5F-G6R; Folprp4 G7F-G8R; Folprp4 G9F-G10R; Folprp4 G11F-G12R and Folprp4 G13F-G14R primers in sequence (j) The PCR product was amplified with Folprp4 cDNA1F-2R as the primer, 1: using cDNA as a template; 2: using hm DNA as a template (k) The PCR product was amplified with Folprp4 cDNA3F-4R as the primer, 1: using cDNA as a template; 2: using hm DNA as a template

Fig.3 Construction for deletion cassette of the Folprp4 M: 1 kb plus DNA Ladder (a) PCR products of amplification of upstream and downstream sequences of Folprp4 gene, 1: PCR products of amplification of upstream sequences of Folprp4 gene; 2: PCR products of amplification of downstream sequences of Folprp4 gene (b) PCR products of amplification of upstream and downstream sequences of hph gene, 1: PCR products of amplification of upstream sequences of hph gene; 2: PCR products of amplification of downstream sequences of hph gene (c) Split-Marker overlapping PCR products of Folprp4 gene, 1: Folprp4 KO1F-HY/R overlapping PCR products of Folprp4 gene; 2: YG/F -Folprp4 KO4R overlapping PCR products of Folprp4 gene

Fig.4 Screening for knockout mutant of Folprp4 M: 1 kb plus DNA Ladder (a) Negative screening of Folprp4 knockout mutants using Folprp4 N1F-N2R as primers (b) Positive screening of Folprp4 knockout mutants using HYG/F-HYG/R as primers (c) Positive screening of Folprp4 knockout mutants using Folprp4 KO1F-HY/R as primers (d) Positive screening of Folprp4 knockout mutants using YG/F-Folprp4 KO4R as primers (b-d) 1-3: Folprp4 transformant DNA was used as the template; 4: hm DNA as template for a positive control; 5: ddH₂O as template for a negative control

Fig.5 Construction of complementary plasmid pZDH1 M: 1 kb plus DNA Ladder (a)1-2: pZWH1 plasmid DNA (b) Linearization of plasmid vector pZWH1 (c)1: BamH I enzyme digestion of pZWH1; 2: EcoR I enzyme digestion of pZWH1 (d) 1-2: The PCR product was amplified using Folprp4 genomic DNA as template and Folprp4 Com1F-Com2R as primer (e) Sma I enzyme digestion target gene fragment

Fig.6 Screening of complementary plasmid pZDH1 M: 1 kb plus DNA Ladder (a) Different strains were used as templates and Folprp4 N1F-N2R as primers for PCR products (b)1-2: Colony 28, 31 plasmid DNA (c) The PCR product was amplified using plasimd DNA as template and NeoF-NeoR as primer (d) The PCR product was amplified using plasimd DNA as template and Folprp4 Com1F-Com2R as primer (e) Sma I enzyme digestion recombinant plasmid pZDH1

Fig.7 Screening of Folprp4-C PCR M: 1 kb plus DNA Ladder (a) 1-3: Transformants 1-3 Folprp4 cDNA 1F-2R PCR screening; 4-5: ΔFolprp4 DNA Folprp4 cDNA 1F-2R PCR control; 6: hm DNA Folprp4 cDNA 1F-2R PCR control; 7: ddH₂O Folprp4 cDNA 1F-2R PCR control (b) 1-3: Transformants 1-3 NeoF-NeoR PCR screening; 4-5: ΔFolprp4 DNA NeoF-NeoR PCR control; 6: hm DNA NeoF-NeoR PCR control; 7: ddH₂O NeoF-NeoR PCR control (c) 1-3: Transformants 1-3 HYG/F -HYG/R PCR screening; 4-5: ΔFolprp4 DNA HYG/F -HYG/R PCR control; 6: hm DNA HYG/F -HYG/R PCR control; 7: ddH₂O HYG/F -HYG/R PCR control

Fig.8 Observation of colony morphology and diameter measurement of hm, ΔFolprp4, ecFolprp4 and Folprp4-C (a, b, d, e) Mycelial morphology on TCC medium at 6 day, the black arrow shows the location of Folprp4 mycelia (c) Mycelial morphology of ΔFolprp4 on TCC medium at 9 day (f) Diameters of hm, ΔFolprp4, ecFolprp4 and Folprp4-C were measured for 7 days

Table 2 Determination of growth rate of different strains

Fig.9 Conidia morphology of hm, ΔFolprp4, ecFolprp4 and Folprp4-C (a~d) The conidia morphology of hm, ΔFolprp4, ecFolprp4 and Folprp4-C were cultured in CMC medium for 25 days

Table 3 Conidia yield of hm, ecFolprp4, Folprp4-C and ΔFolprp4

Fig.10 Infection of flax seedlings by conidia suspension of hm, ΔFolprp4, ecFolprp4 and Folprp4-C (a) Positive view of infection of hm, ΔFolprp4, ecFolprp4 and Folprp4-C conidia suspensions on flax seedlings (b) Top view of infection of hm, ΔFolprp4, ecFolprp4 and Folprp4-C conidia suspensions on flax seedlings


[1]	王政逸, 李德葆. 尖孢镰刀菌的遗传多态性. 植物病理学报, 2000, 30(3):193-199.

[1]	Wang Z Y, Li D B. Genetic diversity in Fusarium oxysporum. Acta Phytopathologica Sinica, 2000, 30(3):193-199.

[2]	Iida Y, Fujiwara K, Yoshioka Y, et al. Mutation of FVS1, encoding a protein with a sterile alpha motif domain, affects asexual reproduction in the fungal plant pathogen Fusarium oxysporum. FEMS Microbiology Letters, 2014, 351(1):104-112. doi: 10.1111/fml.2014.351.issue-1

[3]	Ohara T, Tsuge T. FoSTUA, encoding a basic helix-loop-helix protein, differentially regulates development of three kinds of asexual spores, macroconidia, microconidia, and chlamydospores, in the fungal plant pathogen Fusarium oxysporum. Eukaryotic Cell, 2004, 3(6):1412-1422. doi: 10.1128/EC.3.6.1412-1422.2004

[4]	沈瑞清, 张萍, 郭成瑾, 等. 宁夏镰刀菌属(Fusarium link)真菌的研究. 安徽农业科学, 2012, 40(16):8869-8870, 8875.

[4]	Shen R Q, Zhang P, Guo C J, et al. Study on fungi belonging to Fusarium link in ningxia Hui autonomous region. Journal of Anhui Agricultural Sciences, 2012, 40(16):8869-8870, 8875.

[5]	张志铭, 刘信义, 陈书龙, 等. 亚麻枯萎病菌鉴定. 河北农业大学学报, 1994(2):107.

[5]	Zhang Z M, Liu X Y, Chen S L, et al. Identification of Flax wilt pathogen. Journal of Agricultural University of Hebei, 1994(2):107.

[6]	潘虹, 关凤芝, 吴广文, 等. 尖孢镰刀菌亚麻专化型生物学特性研究. 东北农业大学学报, 2011, 42(7):50-56.

[6]	Pan H, Guan F Z, Wu G W, et al. Study on biological characteristics of Fusarium oxysporum Schl.f.sp.lini. Journal of Northeast Agricultural University, 2011, 42(7):50-56.

[7]	李英, 杜春梅. 致病性尖孢镰刀菌毒力因子的研究进展. 中国农学通报, 2021, 37(12):92-97.

[7]	Li Y, Du C M. Virulence factors of pathogenic Fusarium oxysporum: research progress. Chinese Agricultural Science Bulletin, 2021, 37(12):92-97.

[8]	叶旭红, 林先贵, 王一明. 尖孢镰刀菌致病相关因子及其分子生物学研究进展. 应用与环境生物学报, 2011, 17(5):759-762.

[8]	Ye X H, Lin X G, Wang Y M. Progress in research on pathogenic factors and related molecular biology of Fusarium oxysporum. Chinese Journal of Applied and Environmental Biology, 2011, 17(5):759-762.

[9]	Kim H S, Park S Y, Lee S, et al. Loss of cAMP-dependent protein kinase A affects multiple traits important for root pathogenesis by Fusarium oxysporum. Molecular Plant-Microbe Interactions, 2011, 24(6):719-732. doi: 10.1094/MPMI-11-10-0267

[10]	Staley J P, Woolford J L Jr. Assembly of ribosomes and spliceosomes: complex ribonucleoprotein machines. Current Opinion in Cell Biology, 2009, 21(1):109-118. doi: 10.1016/j.ceb.2009.01.003

[11]	Banroques J, Abelson J N. PRP4: a protein of the yeast U4/U6 small nuclear ribonucleoprotein particle. Molecular and Cellular Biology, 1989, 9(9):3710-3719. doi: 10.1128/mcb.9.9.3710-3719.1989 pmid: 2528687

[12]	Gao X L, Zhang J, Song C N, et al. Phosphorylation by Prp4 kinase releases the self-inhibition of FgPrp31 in Fusarium graminearum. Current Genetics, 2018, 64(6):1261-1274. doi: 10.1007/s00294-018-0838-4

[13]	Bitencourt T A, Oliveira F B, Sanches P R, et al. The prp4 kinase gene and related spliceosome factor genes in Trichophyton rubrum respond to nutrients and antifungals. Journal of Medical Microbiology, 2019, 68(4):591-599. doi: 10.1099/jmm.0.000967 pmid: 30900975

[14]	Schwelnus W, Richert K, Opitz F, et al. Fission yeast Prp4p kinase regulates pre-mRNA splicing by phosphorylating a non-SR-splicing factor. EMBO Reports, 2001, 2(1):35-41. doi: 10.1093/embo-reports/kve009 pmid: 11252721

[15]	Luetzelberger M, Kufert N F. The Prp4 kinase: its substrates, function and regulation in pre-mRNA splicing//Cai H. Protein Phosphorylation in Human Health. Croatia: InTech, 2012:195-216.

[16]	Schneider M, Hsiao H H, Will C L, et al. Human PRP4 kinase is required for stable tri-snRNP association during spliceosomal B complex formation. Nature Structural & Molecular Biology, 2010, 17(2):216-221. doi: 10.1038/nsmb.1718

[17]	Gross T, Lützelberger M, Weigmann H, et al. Functional analysis of the fission yeast Prp4 protein kinase involved in pre-mRNA splicing and isolation of a putative mammalian homologue. Nucleic Acids Research, 1997, 25(5):1028-1035. doi: 10.1093/nar/25.5.1028 pmid: 9102632

[18]	Ahmed M B, Islam S U, Sonn J K, et al. PRP4 kinase domain loss nullifies drug resistance and epithelial-mesenchymal transition in human colorectal carcinoma cells. Molecules and Cells, 2020, 43(7):662-670.

[19]	Wang C F, Zhang S J, Hou R, et al. Functional analysis of the kinome of the wheat scab fungus Fusarium graminearum. PLoS Pathogens, 2011, 7(12):e1002460. doi: 10.1371/journal.ppat.1002460

[20]	Michielse C B, Rep M. Pathogen profile update: Fusarium oxysporum. Molecular Plant Pathology, 2009, 10(3):311-324. doi: 10.1111/j.1364-3703.2009.00538.x pmid: 19400835

[21]	Catlett N L, Lee B N, Yoder O C, et al. Split-marker recombination for efficient targeted deletion of fungal genes. Fungal Genetics Reports, 2003, 50(1):9-11. doi: 10.4148/1941-4765.1150

[22]	彭海丽, 侯占铭. 小麦赤霉菌FGSG_04871基因敲除及功能研究. 内蒙古师范大学学报(自然科学汉文版), 2017, 46(3):394-400.

[22]	Peng H L, Hou Z M. Research on knockout and function of FGSG_04871 gene in Fusarium graminearum. Journal of Inner Mongolia Normal University (Natural Science Edition), 2017, 46(3):394-400.

[23]	Gao X L, Jin Q J, Jiang C, et al. FgPrp4 kinase is important for spliceosome B-complex activation and splicing efficiency in Fusarium graminearum. PLoS Genetics, 2016, 12(4):e1005973. doi: 10.1371/journal.pgen.1005973

[24]	Rosenberg G H, Alahari S K, Käufer N F. prp4 from Schizosaccharomyces pombe, a mutant deficient in pre-mRNA splicing isolated using genes containing artificial introns. Molecular & General Genetics: MGG, 1991, 226(1-2):305-309.

[25]	秦岭, 李晓雯, 李丁, 等. 蛋白激酶Cla4对黄曲霉生长发育、毒素合成和致病力的影响. 菌物学报, 2021, 40(1):174-188.

[25]	Qin L, Li X W, Li D, et al. Protein kinase Cla4 regulates morphology development, aflatoxin biosynthesis and pathogenicity of Aspergillus flavus. Mycosystema, 2021, 40(1):174-188.

[26]	Ding S L, Mehrabi R, Koten C, et al. Transducin beta-like gene FTL1 is essential for pathogenesis in Fusarium graminearum. Eukaryotic Cell, 2009, 8(6):867-876. doi: 10.1128/EC.00048-09

[27]	佟强. 禾谷镰孢菌转录调控因子FgCrz1A的鉴定和功能研究. 合肥: 安徽农业大学, 2018.

[27]	Tong Q. Identification and functional characterization of the transcription factor FgCrz1A in Fusarium graminearum. Hefei: Anhui Agricultural University, 2018.

[28]	周欣, 云英子, 郭谱胜, 等. 尖孢镰刀菌番茄专化型中APSES转录因子FolStuA的功能分析. 分子植物育种, 2018, 16(3):772-780.

[28]	Zhou X, Yun Y Z, Guo P S, et al. Functional analysis of APSES transcription factor FolStuA in Fusarium oxysporum f.sp. lycopersici. Molecular Plant Breeding, 2018, 16(3):772-780.

[29]	孙玲, 褚小静, 郝宇, 等. 尖孢镰刀菌FoPLC4参与调控孢子形成和致病性. 中国农业科学, 2014, 47(12):2357-2364.

[29]	Sun L, Chu X J, Hao Y, et al. FoPLC4, encoding phospholipase C4, is involved in sporulation and pathogenicity in Fusarium oxysporum. Scientia Agricultura Sinica, 2014, 47(12):2357-2364.

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